Non-destructive techniques for in situ analysis of samples whose chemical and compositional properties vary with position in three dimensions have many applications in the physical and biological sciences as well as in industrial process monitoring and quality control. In medical imaging, for example, 3-D spectrochemical images can be used for noninvasive in situ visualization, identification and discrimination between different types of healthy tissue and between healthy and diseased tissues. Clearly, this capability would be of great benefit to both patient and physician because it would aid in the rapid diagnosis of a potentially life threatening condition (e.g. benign versus cancerous tumors) in a manner less invasive than present biopsy techniques.
Analysis of spectra of materials throughout the electromagnetic spectrum, and, particularly in the optical and infrared portions of the spectrum, enables molecular species identification and provides information on molecular structure. Different parts of the spectrum are particularly suited to specific inquiries, as well known in the spectroscopic arts. For example, the structure of molecular rotation, molecular vibration, and electronic structure are typically characteristic of successively shorter spectroscopic regimes, from the far infrared to the visible. Other spectroscopic techniques, such as Raman scattering and fluorescence, wherein the wavelength of excitation differs from the wavelength of interrogation, are included within the understanding of "spectroscopy" in all instances used herein and in the appended claims.
Spectra of two-dimensional scenes are routinely obtained and analyzed. Several techniques are also known for acquiring three-dimensional (3-D) spectrochemical images. These include optical tomography, solution of the inverse photon scattering problem, confocal scanning optical microscopy, and deconvolution microscopy.
Optical tomography requires illumination of a body from a plurality of angles, with a camera moved in tandem with the source of illumination in order to record a series of 2-D images at different angular positions. Tomographic algorithms are then applied to the acquired data set to reconstruct a 3-D image of the illuminated volume. Optical tomography has been successful in 3-D reconstruction of visible light absorption and fluorescence images. The necessity to make many angularly-spaced optical transmission measurements on an isolate specimen makes this approach slow and prevents its usage where there is limited access to the specimen (such as inside the body).
In the inverse scattering approach, a consistent solution is sought to a series of spatially-resolved time or frequency domain measurements to recover the 3-D spectroscopic image. Mostly applicable to 3-D spectrochemical imaging through a highly scattering medium, this technique has been demonstrated with some success for acquisition of 3-D fluorescence and near infrared (NIR) absorption spectroscopic images. The drawbacks of this approach include the non-uniqueness and instability of the solutions obtained and the computational-intensivity of the technique.
A third approach utilizes the inherent spatial discrimination properties of the confocal scanning optical microscope (CSOM). The CSOM combines focused illumination with spatially filtered detection of the back (or forward) scattered optical wave to detect light that originates from a small volume element (voxel) within the sample and attenuate light from out-of-focus. Moving the sample relative to the voxel and recording either the absorption, fluorescence or Raman spectrum of light passed through the pinhole spatial filter enables construction of a 3-D spectrochemical image of the scanned sample volume.
A fourth option entails measuring the 3-D point spread function (PSF) characterizing a particular optical imaging system (such as a conventional optical microscope), recording a series of axially-spaced 2-D-spectroscopic images with the imaging system, and recovering the original 3-D object by numerical deconvolution of the PSF from the 3-D image stack. This approach has been applied to a variety of spectroscopic image modalities including fluorescence, Raman and absorption spectroscopies. This approach suffers from the well-known numerical limitations of present deconvolution routines.